case report a turner syndrome patient carrying a mosaic...

Case ReportA Turner Syndrome Patient Carrying a Mosaic DistalX Chromosome Marker

Roberto L. P. Mazzaschi,1 Juliet Taylor,2 Stephen P. Robertson,3

Donald R. Love,1,4 and Alice M. George1

1 Diagnostic Genetics, LabPlus, Auckland City Hospital, P.O. Box 110031, Auckland 1148, New Zealand2Genetic Health Service New Zealand-Northern Hub, Auckland City Hospital, Private Bag 92024, Auckland 1142, New Zealand3Department of Paediatrics and Child Health, Dunedin School of Medicine, University of Otago, P.O. Box 913,Dunedin 9054, New Zealand

4 School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

Correspondence should be addressed to Alice M. George; [email protected]

Received 31 December 2013; Accepted 5 February 2014; Published 17 March 2014

Academic Editors: M. Fenger, G. Vogt, and X. Wang

Copyright © 2014 Roberto L. P. Mazzaschi et al.This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in anymedium, provided the originalwork is properly cited.

A skin sample from a 17-year-old female was received for routine karyotyping with a set of clinical features including clonicseizures, cardiomyopathy, hepatic adenomas, and skeletal dysplasia. Conventional karyotyping revealed amosaic Turner syndromekaryotype with a cell line containing a small marker of X chromosome origin.This was later confirmed on peripheral blood culturesby conventional G-banding, fluorescence in situ hybridisation andmicroarray analysis. Similar Turnermosaicmarker chromosomecases have been previously reported in the literature, with a variable phenotype ranging from the mild “classic” Turner syndrometo anencephaly, agenesis of the corpus callosum, complex heart malformation, and syndactyly of the fingers and toes. This casereport has a phenotype that is largely discordant with previously published cases as it lies at the severe end of the Turner variantphenotype scale.The observed cytogenetic abnormalities in this study may represent a coincidental finding, but we cannot excludethe possibility that the marker has a nonfunctioning X chromosome inactivation locus, leading to functional disomy of those genescarried by the marker.

1. Introduction

Turner syndrome (TS) presents with a characteristic mildphenotype with some degree of variability [1].Themajority ofpatients have short stature, are infertile, and do not developsecondary sexual characteristics. Less consistent abnormal-ities include webbed neck, renal malformations (>50%),and cardiac defects (10%), while intelligence is considerednormal. Approximately three-quarters of TS females inherittheir X chromosome maternally [2].

Turner syndrome mosaics are also well documented andcan be subcategorised according to whether the second cellline contains a whole or part of a sex chromosome. Jacobs etal. (1997) showed that, of 84 Turner syndrome cases with astandard karyotype of 45,X, 16% were mosaic, with a secondcell line containing a ring X chromosome (45,X/46,X,r(X))[3]. The phenotypic variability of these mosaics is largely

dependent on the size of the ring and the presence of afunctioning XIST.

XIST is a cis-acting gene in the X-inactivation centre(XIC), located in band Xq13. As a general rule, when oneX chromosome has an imbalance that does not involvean autosome, the XIC on the abnormal X chromosomeis activated. This activation leads to nonrandom skewingof X chromosome inactivation, with the XIST transcriptinactivating the abnormal chromosome. The phenotype ofthis group of patients is generally that of amild Turner variantphenotype [2].

Marker or ring X chromosomes (r(X)) lacking a func-tional XIST confer functional disomy of the duplicatedregion, which is expected to lead to a more severe phenotype.With the exception of mental retardation/developmentaldelay, they also share little phenotypic concordance. Migeonet al. (2000) described two TS variant mosaic cases with large

Hindawi Publishing CorporationCase Reports in GeneticsVolume 2014, Article ID 597314, 5 pageshttp://dx.doi.org/10.1155/2014/597314

2 Case Reports in Genetics

imbalances and intact XIST regions on their markers [4].These patients presented withmental retardation and fall intothe category of “tiny ring X syndrome.”

The mechanism for generating ring chromosomes isthought to be initiated by two chromosome breakage eventsoccurring at either side of a centromere, followed by fusionof the two broken ends of the centromere-containing frag-ment [1]. This event is thought to occur at meiosis. Non-ring marker chromosomes require the additional step oftelomere addition/formation at the broken ends. Ring chro-mosomes bring the added complication of “ring chromosomesyndrome,” whereby concentric rings are generated at celldivision, with the inevitable nondisjunction, leading to morethan one copy being present in some cells (also referred to asdynamic mosaicism).

Here we report a case which, from a cytogenetic view-point, appears to represent a relatively straightforward exam-ple of a Turner syndrome variant mosaic karyotype, with asmall marker chromosome of X chromosome origin. How-ever, from a clinical perspective, this case study has a rathersevere phenotype that does not fit with patients previouslyreported in the literature. The final karyotype was based ona combination of FISH analysis, as well as conventional (G-banded) and molecular (microarray) karyotyping.

2. Case Report

The proband presented at nine months of age with poorgrowth/failure to thrive (below the 3rd percentile for herweight, length, and head circumference) and global devel-opmental delay. There followed a lengthy period of deteri-oration, with additional problems including type I diabetes(at 10 years of age), clonic seizures, cardiomyopathy, hepaticadenomas, and skeletal dysplasia. Prior to initial karyotyping,a DNA sample was sent for entire mitochondrial DNAgenome sequencing, which did not identify any pathogenicmutations. The gene for Wolcott-Rallison syndrome (WRS),EIF2AK3, was also sequenced but returned a negative result.

At 22 years of age she was assessed as functioning at thelevel of a 5-year-old child. She had delayed secondary sexualcharacteristic development and now presents with partialovarian failure and growth hormone deficiency. The medicalhistory of the rest of the family provided no additionalinformation other than the proband having a brother withAsperger syndrome.

Three long-term closed flask fibroblast explant cultureswere set up according to the protocol adapted from Rooney(2001) [5] when the proband was 17 years of age. G-bandedchromosome preparations (at a resolution of 400 bands perhaploid set) were made following the Seabright protocol[6]. 20/35 (57%) cells had a 45,X karyotype with a singleX chromosome while the remaining cells had 46 chromo-somes, with a single X chromosome and an additional smallmarker chromosome (possibly a ring), of unknown origin.Additional fluorescence in situ hybridisation testing wascarried out on interphase nuclei of subcultured fibroblastsusing the (Vysis) Aneuscreen FISH probe set (cenX (DXZ1),cenY (DYZ3), 13q14 (RB1), cen18 (D18Z1), and 21q22.13-q22.2 (D21S259/D21S341/D21S342)). In the case ofmosaicism

studies, a larger number of interphase nuclei were scored (atleast 200), compared to our standard analysis of a minimumof 50 nuclei per probe set. The result was consistent witha female karyotype with a single X chromosome (Turnersyndrome) in most nuclei, with a low-level two-copy Xcentromere signal pattern seen in 8/214 nuclei. The apparentdiscordance between the G-banded karyotype and later FISHanalysis may be due to random loss of the marker cell linethrough continued subculturing.

A peripheral blood sample in lithiumheparin was recom-mended at a higher chromosome band resolution (550 bandsper haploid set) for further characterisation of the marker.G-banded (conventional) karyotype analysis on peripheralblood synchronised cultures using a modified method ofGallo et al. [7] confirmed the fibroblast culture findings,with 16/30 (53%) cells having a single X chromosome and14/30 (47%) having the additional marker chromosome ofunknown origin. FISH studies using the Vysis Xcen (DXZ1),Ycen (DYZ3), and Yp11.3 (SRY) probes onmetaphase spreadswere also carried out (Figure 1). The Xcen probe of cen-tromeric alpha satellite DNA covered the region Xp11.1-Xq11.1and hybridised to the normal X chromosome centromere andthe marker in 9/30 (30%) cells. The remaining 21/30 (70%)cells, which lacked the marker, only showed hybridisation tothe X chromosome centromere. This was considered to bea more reliable result than the previous interphase FISH ofcultured fibroblast cells. The proportion of the two cell linesshowed concordance with the G-banded karyotype results ofboth tissue types. No hybridisation of Y centromere or SRYprobes were detected on the marker or other chromosomes.Parental blood samples were requested but never received.

Five years later an EDTA peripheral blood sample wasreceived for molecular karyotyping as previously described[8]. Microarray analysis was carried out on extracted DNAusing the Affymetrix Cytogenetics Whole Genome 2.7MArray, Affymetrix Chromosome Analysis Suite (ChAS)v1.0.1/na30.1. An abnormal female mosaic molecular kary-otype was determined as arr[hg18] Xq11.1q21.1(61,934,835-78,510,961)x1∼2. This result indicated the presence of twogenotypes: one with a single X chromosome complement,with no Y chromosome, and another of a single X chromo-some complement with a 16.6Mb duplication of X chromo-some material from Xq11.1q21.1 (Figure 2), again with no Ychromosome.

An estimate of the level of mosaicism could not bemade based on the microarray data. Taken together, the datasuggest a mosaic marker chromosome comprised of an Xcentromere (from FISH) and pericentromeric euchromatinfrom the long arm of the X chromosome, including the Xinactivation locus XIST. Due to the limits of the microar-ray assay (there is no probe coverage for centromeres ortelomeres), the finding of X centromeric hybridisation to themarker detected on metaphase FISH could not be confirmedby molecular karyotyping.

Finally, molecular X chromosome inactivation analysiswas attempted in order to determine if the markerchromosome was being expressed. If the marker wasactive, functional disomy for the included genes couldprovide an explanation for the observed severe phenotype.

Case Reports in Genetics 3

(a) (b)

Figure 1: FISH analysis of the proband’s cells. (a) The X chromosome centromere probe (Spectrum Green; DXZ1) on an inverted grey scaleDAPI-stained metaphase spread shows hybridisation to both the normal X homologue and the marker chromosome. (b) X chromosomecentromere probe (Spectrum Green; DXZ1) with a chromosome 18 centromere probe (Aqua; D18Z1) as a control. 1-2 X centromere signalsper interphase nucleus can be seen.

p22.2 21.1 11. 1 Xq21.1 q22.3 Xq23 24 Xq25 Xq28ChrX (q11.1q21.1)

(a)

ScalechrX:

hg1865,000,000 70,000,000 75,000,000

OMIM genes, dark green, are disease-causing

Refseq genes

300429300647300891

300889309845

300694300353

300167

300276313700

300127

300689300035

300420

300451300714300139

300038301770300632300521

300189300311

300443

300883300033

308380300188300336

304040300061300084300332

313650

300452300255

300369300574

300252300687312760300149300269

311870300878

300026

300025300181314670

300832300095

300379

300524

300135300656

300576

300760300759

300827300032

300715300885300011300567

311800300754

300201

300086300529

300222

SPIN4LOC92249ARHGEF9ARHGEF9ARHGEF9

MIR1468AMER1

ASB12

MTMR8

ZC4H2ZC4H2ZC4H2ZC4H2ZC4H2

ZC3H12B

LAS1LLAS1LLAS1L

FRMD8P1

MSNMIR223

VSIG4VSIG4VSIG4

VSIG4VSIG4

HEPHHEPHHEPH

EDA2R

EDA2REDA2R

ARAROPHN1

YIPF6YIPF6

STARD8STARD8STARD8

EFNB1PJA1PJA1

PJA1FAM155B

EDA

EDAEDAEDAEDA

MIR676

AWAT2

OTUD6A

IGBP1

DGAT2L6

AWAT1

P2RY4ARR3

RAB41PDZD11

KIF4A

GDPD2

GDPD2GDPD2GDPD2

DLG3DLG3DLG3TEX11TEX11

SLC7A3SLC7A3

SNX12SNX12SNX12SNX12SNX12

FOXO4

FOXO4

CXorf65CXorf65

IL2RG

MED12NLGN3

NLGN3NLGN3

GJB1

GJB1ZMYM3ZMYM3

ZMYM3ZMYM3

NONONONONONONONO

ITGB1BP2

TAF1TAF1

INGX

OGTOGT

ACRCCXCR3CXCR3CXorf49

CXorf49BCXorf49B

CXorf49FLJ46446

LOC100132741

NHSL2

RPS26P11

RGAG4

FLJ44635

PIN4PIN4PIN4

ERCC6L

RPS4XCITED1CITED1CITED1

CITED1HDAC8HDAC8HDAC8

HDAC8HDAC8

HDAC8HDAC8PHKA1PHKA1PHKA1

FAM226AFAM226B

LINC00684DMRTC1

DMRTC1BDMRTC1

DMRTC1BLINC00684

FAM226BFAM226A

PABPC1L2BPABPC1L2A

NAP1L6

NAP1L2CDX4

MAP2K4P1CHIC1

TSIXXIST

JPX

FTX

MIR374BMIR374C

MIR545MIR374AZCCHC13

SLC16A2

RLIM

RLIM

KIAA2022

ABCB7

ABCB7ABCB7ABCB7

ABCB7

UPRT

UPRTZDHHC15ZDHHC15ZDHHC15

TTC3P1

MAGEE2

PBDC1

MAGEE1MIR384

FGF16

ATRXATRX

MAGT1

COX7BATP7APGAM4

PGK1TAF9B

CYSLTR1

ZCCHC5LPAR4LPAR4

MIR4328P2RY10

P2RY10GPR174

ITM2AITM2A

5MbHuman Mar. 2006 (NCBI36/hg18) chrX: 61,934,835–78,510,961(16,576,127 bp)

(b)

Figure 2: Schematic of the chromosome X region comprising the marker X chromosome (a) shows an ideogram of chromosome X, togetherwith the region of the marker chromosome. (b) shows the OMIM and Refseq genes that lie on the marker chromosome.These graphics weretaken from the UCSC genome browser (http://genome.ucsc.edu/).

4 Case Reports in Genetics

Unfortunately, X chromosome inactivation could notbe assessed due to the lack of informativeness at the Xchromosome amelogenin locus.

3. Discussion

In the case of a suspicion of Turner syndrome, a stan-dard G-banded karyotype analysis is usually requested ona peripheral blood culture. Interestingly, the case reportedhere was not referred to confirm Turner syndrome but toconfirm/excludeWolcott-Rallison syndrome, which involveda number of metabolic and genetic tests.

Combining the conventional karyotyping and FISHdata together with molecular karyotyping has allowed forthe full characterisation of the genetic content of themarker chromosome. This marker contained many genes,including twenty-four classed by OMIM (Online MendelianInheritance in Man; http://www.ncbi.nlm.nih.gov/omim) asdisease-causing. Of these, OPHN1, IGBP1, DLG3, NLGN3,and ZDHHC15 are associated with mental retardation orAsperger syndrome phenotypes. Bedeschi et al. [9] describeda case of a male with a duplication on the X chromosomefrom Xq12 to Xq13.1, a region that includes the OPHN1gene. He had severe mental retardation but an otherwisediscordant phenotype compared to the case described here.This represents a much smaller duplication than our casestudy and was also nonmosaic but is another example offunctional disomy of a part of the X chromosome. Hemmatet al. [10] described a rare case of an acentric marker Xchromosome containing an activated neocentromere distal tothis case study, at Xq21.2. This example raises the possibilityof a similar sequence (of DNA with a degree of homology tocentromeric sequences) being present in the marker of thecase described here, which has been forced into activation togive it stability in cell division. This suggestion is compatiblewith the observed cytogenetic findings.

Migeon et al. [4] described two TS variant mosaic casessimilar in appearance to our case study, but with largerimbalances, with intact XIST regions on their markers.They presented with mental retardation but an otherwisediscordant severe phenotype to the case study reported here.These, as with most small r(X) cases in the literature, werepublished before microarray analysis was available, makinggene content comparisons with this case difficult. It shouldalso be noted that the marker chromosome is described asa ring based only on its G-banded appearance. However,telomere FISH studies have not been carried out, and so thepossibility that the marker has telomeres, and is not a ring,cannot be excluded.

SNP (Single Nucleotide Polymorphism) analysis (datanot shown) revealed homozygosity along the entire lengthof the X chromosome, including the region of disomy (themarker chromosome). Heterozygosity would have indicatedthe involvement of a second nonhomologous X chromosome(presumably from the other parent), in the formation ofthe marker. While not conclusive, our data suggest that themarker X may well have been derived from the normal Xchromosome already present, rather than from a secondchromosome X homologue.

Ring chromosomes also raise the possibility of “ringchromosome syndrome” occurring. At the DNA duplicationphase of the cell cycle, concentric rings can accidentallybe generated prior to cell division, with the inevitablenondisjunction leading to more than one copy of the ringsegregating into some cells, which is also referred to asdynamicmosaicism. However, there was no evidence of “ringchromosome syndrome” in the G-banded analysis or FISHresults. For non-ring marker chromosome formation, theadditional steps of telomere generation and capping need tooccur at the broken ends of the forming marker in order toconfer chromosome stability.

For the purposes of this discussion, the mosaic markercan be considered as a 16.6Mb gain of X chromosomematerial (from Xq11.1q21.1), against a Turner syndromegenotype background. As already discussed, the pheno-typic effect of genes on the marker would only apply ifa nonfunctioning copy of XIST was present. The markerdid contain many genes, including twenty-four classed byOMIM as disease-causing (Online Mendelian Inheritancein Man (OMIM); http://www.ncbi.nlm.nih.gov/omim). Ofthese, OPHN1, IGBP1, DLG3, NLGN3, and ZDHHC15 areassociated with mental retardation or Asperger syndromephenotypes. Bedeschi et al. [9] described a case of a malewith a duplication on the X chromosome from Xq12 toXq13.1, a region including the OPHN1 gene. He had severemental retardation, but an otherwise discordant phenotypecompared to the case study. This is included here as itrepresents an example of functional disomy for the regionof the X chromosome under investigation (although thereported case was much smaller, nonmosaic, and in amale).

Turner syndrome variants include female individualswith partial deletions in the “p” and/or “q” arms of oneX chro-mosome. Deletions of certain X chromosome regions/genescan lead to specific phenotypic features which are char-acteristic of “full” or “classic” Turner syndrome. Deletionsof the SHOX gene, located in the PAR (pseudoautosomalregion) at Xp22.33, are associated with short stature althoughthis characteristic TS feature is not noted in the case study.Primary ovarian failure (POF) has been associated withdeletions of the FMR1 gene (POF1) at Xq26–q28 [11] and theDIAPH2 gene (POF2A) at Xq21.33 [12]. Type I diabetes hasalso been linked to the Turner syndrome phenotype [13].

The difficulty presented to the genetic counselors inthis case was in trying to correlate the cytogenetic findingswith the patient’s phenotype. This patient was thought torepresent an example of the severe end of the Turner syn-drome spectrum, in tandem with poorly controlled diabetes.Hepatic adenomas had been previously reported in a childwith Turner syndrome on growth hormone supplementation[14]. The karyotypic findings were considered to provide aunifying diagnosis for the patient’s multiple comorbidities.The parents of this case study have not been karyotyped,making it impossible to give a risk of recurrence for futurepregnancies. Similarly, the presence of this ring in a parentcould also provide more information concerning a genotype-phenotype correlation. Transmission of ring X chromosomeshas been previously described in both male and female

Case Reports in Genetics 5

offspring. However, although all these cases involved non-supernumerary chromosomes, the rings were substantial insize with breakpoints more distal than those seen in this case[1].

4. Conclusions

This case study represents the coincidental finding of anindividual with a severe set of clinical abnormalities and aTurner syndromemosaic karyotype.The cytogenetic findingscan be used to account for some of the observed phenotypicfeatures, but the paucity of similar cases published in theliterature makes a genotype-phenotype correlation difficult.We consider it likely that our patient’s observed severephenotype is due to functional disomy for those genes carriedon the marker chromosome.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

References

[1] R. J. M. Gardner, G. R. Sutherland, and L. G. Shaffer, Chromo-some Abnormalities and Genetic Counseling, Oxford UniversityPress, New York, NY, USA, 4th edition, 2011.

[2] A. Schinzel, Catalogue of Unbalanced Chromosome Aberrationsin Man, De Gruyter, Berlin, NY, USA, 2nd edition, 2001.

[3] P. Jacobs, P. Dalton, R. James et al., “Turner syndrome: acytogenetic and molecular study,” Annals of Human Genetics,vol. 61, no. 6, pp. 471–483, 1997.

[4] B. R. Migeon, M. Ausems, J. Giltay et al., “Severe phenotypesassociated with inactive ring X chromosomes,” The AmericanJournal of Medical Genetics, vol. 93, no. 1, pp. 52–57, 2000.

[5] D. E. Rooney, Human Cytogenetics Constitutional Analysis,Oxford University Press, New York, NY, USA, 3rd edition, 2001.

[6] M. Seabright, “A rapid banding technique for human chromo-somes,”The Lancet, vol. 2, no. 7731, pp. 971–972, 1971.

[7] J. H. Gallo, J. V. Ordonez, G. E. Brown, and J. R. Testa,“Synchronization of human leukemic cells: relevance for high-resolution chromosome banding,”Human Genetics, vol. 66, no.2-3, pp. 220–224, 1984.

[8] A. Al-Murrani, F. Ashton, S. Aftimos, A. M. George, and D.R. Love, “Amino-terminal microdeletion within the CNTNAP2gene associated with variable expressivity of speech delay,” CaseReports in Genetics, vol. 2012, Article ID 172408, 4 pages, 2012.

[9] M. F. Bedeschi, A. Novelli, L. Bernardini et al., “Associationof syndromic mental retardation with an Xq12q13.1 duplicationencompassing the oligophrenin 1 gene,” The American Journalof Medical Genetics, Part A, vol. 146, no. 13, pp. 1718–1724, 2008.

[10] M. Hemmat, B. T. Wang, P. E. Warburton et al., “Neocentric X-chromosome in a girl with Turner-like syndrome,” MolecularCytogenetics, vol. 5, no. 1, article 29, 2012.

[11] T. Eggermann, D. Meschede, H. Schuler et al., “Prematureovarian failure associated with a small terminal Xq deletion:narrowing the POF1 region down to Xq27.2/Xq27.3-qter,” Clini-cal Genetics, vol. 67, no. 5, pp. 434–437, 2005.

[12] A. Marozzi, E. Manfredini, M. Tibiletti et al., “Moleculardefinition of Xq common-deleted region in patients affected by

premature ovarian failure,” Human Genetics, vol. 107, no. 4, pp.304–311, 2000.

[13] M. Callea, F. Radovich, M. Cappa, and G. Clarich, “Turner’ssyndrome with mental retardation, microcephaly and type 1diabetes in a 6 year old child: case report and literature review,”Minerva Pediatrica, vol. 65, no. 2, pp. 251–252, 2013.

[14] R. A. Morotti, M. Killackey, B. L. Shneider, A. Repucci, S. Emre,and S. N. Thung, “Hepatocellular carcinoma and congenitalabsence of the portal vein in a child receiving growth hormonetherapy for Turner syndrome,” Seminars in Liver Disease, vol. 27,no. 4, pp. 427–431, 2007.

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